A 3-day delay in synovial fluid crystal identification did not hinder the reliable detection of monosodium urate and calcium pyrophosphate crystals.

BACKGROUND: Arthrocentesis is an essential emergency step in managing patients with acute arthritis. To identify a bacterial infection, Gram staining is performed promptly. However, crystal analysis may not be immediately performed in many facilities. Being considered not to be stable over time, synovial fluid (SF) is sometimes discarded instead of being stored for crystal identification. OBJECTIVE: The aim of this study was to assess the detectability of monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals in SF over a period of 3 days. METHODS: Consecutive SF samples from 75 joints were analyzed for MSU, CPP crystals, and pH. Two independent observers evaluated the samples by regular light and polarization microscopy immediately after arthrocentesis and after 1, 2, and 3 days at room temperature or at 4°C. RESULTS: Of 75 samples, 27 contained crystals (16 MSU, 6 CPP, 5 both); semiquantitative counts of both MSU and CPP crystals did not change significantly after 3 days. There was no new formation of crystals in any of the crystal-negative samples, which was independent of the storage temperature. Synovial fluid pH was not predictive of crystals and did not change over time. CONCLUSIONS: Although immediate workup for microbiology, including Gram stain and culture, is indispensable and well established, crystal analysis may at times not be immediately performed. Our study suggests that when crystal identification cannot be done immediately, it can be safely performed up to 3 days after arthrocentesis when SF is stored at 4°C or even at stable room temperature (20°C).

Glycitein decreases the generation of murine osteoclasts and increases apoptosis.

BACKGROUND: Phytoestrogens, especially genistein, have been shown to have bone beneficial effects in vitro and in vivo. However, the effect of glycitein on bone cells is not known. The aim of this study was to investigate the effects of glycitein on osteoclast differentiation and apoptosis in vitro. METHODS: Bone marrow-derived osteoclasts were cultured with various concentrations (0.01-100 nM) of glycitein. Osteoclast generation was assessed by the number of multinucleated, tartrate-resistant acid phosphatase (TRAP)-positive cells, and apoptosis by the activity of caspase 3/7. Bone-marrow-derived osteoblasts were cultured in the presence of 10 nM glycitein. Subsequently, gene expression levels of receptor activator of NFκB ligand (RANKL), osteoprotegerin (OPG), and interleukin-6 (IL-6) were determined by real-time PCR. RESULTS: Osteoclast generation was inhibited by glycitein in a biphasic-dose-dependent manner and showed the greatest inhibitory effects at 10 nM (-70%, p < 0.01). Glycitein increased caspase 3/7 activity by 15% at a concentration of 10 nM (p < 0.001). Further, 10 nM glycitein significantly decreased the expression of IL-6 (-53%, p < 0.05) and RANKL (-64%, p < 0.05) in osteoblasts but did not change mRNA levels of OPG. CONCLUSIONS: Our data demonstrate that glycitein suppresses osteoclast generation and induces osteoclast apoptosis in vitro to a similar extent as genistein and therefore suggests that glycitein may also exert bone beneficial effects in vivo.

Regulation of sclerostin in glucocorticoid-induced osteoporosis (GIO) in mice and humans.

Glucocorticoids (GC) are used for the treatment of inflammatory diseases, including various forms of arthritis. However, their use is limited, amongst others, by adverse effects on bone. The Wnt and bone formation inhibitor sclerostin was recently implicated in the pathogenesis of GC-induced osteoporosis. However, data are ambiguous. The aim of this study was to assess the regulation of sclerostin by GC using several mouse models with high GC levels and two independent cohorts of patients treated with GC. Male 24-week-old C57BL/6 and 18-week-old DBA/1 mice exposed to GC and 12-week-old mice with endogenous hypercortisolism displayed reduced bone formation as indicated by reduced levels of P1NP and increased serum sclerostin levels. The expression of sclerostin in femoral bone tissue and GC-treated bone marrow stromal cells, however, was not consistently altered. In contrast, GC dose- and time-dependently suppressed sclerostin at mRNA and protein levels in human mesenchymal stromal cells, and this effect was GC receptor dependent. In line with the human cell culture data, patients with rheumatoid arthritis (RA, n = 101) and polymyalgia rheumatica (PMR, n = 21) who were exposed to GC had lower serum levels of sclerostin than healthy age- and sex-matched controls (-40%, P < 0.01 and -26.5%, P < 0.001, respectively). In summary, sclerostin appears to be differentially regulated by GC in mice and humans as it is suppressed by GCs in humans but is not consistently altered in mice. Further studies are required to delineate the differences between GC regulation of sclerostin in mice and humans and assess whether sclerostin mediates GC-induced osteoporosis in humans.

Thy-1 (CD90) promotes bone formation and protects against obesity.

Osteoporosis and obesity result from disturbed osteogenic and adipogenic differentiation and present emerging challenges for our aging society. Because of the regulatory role of Thy-1 in mesenchyme-derived fibroblasts, we investigated the impact of Thy-1 expression on mesenchymal stem cell (MSC) fate between osteogenic and adipogenic differentiation and consequences for bone formation and adipose tissue development in vivo. MSCs from Thy-1-deficient mice have decreased osteoblast differentiation and increased adipogenic differentiation compared to MSCs from wild-type mice. Consistently, Thy-1-deficient mice exhibited decreased bone volume and bone formation rate with elevated cortical porosity, resulting in lower bone strength. In parallel, body weight, subcutaneous/epigonadal fat mass, and bone fat volume were increased. Thy-1 deficiency was accompanied by reduced expression of specific Wnt ligands with simultaneous increase of the Wnt inhibitors sclerostin and dickkopf-1 and an altered responsiveness to Wnt. We demonstrated that disturbed bone remodeling in osteoporosis and dysregulated adipose tissue accumulation in patients with obesity were mirrored by reduced serum Thy-1 concentrations. Our findings provide new insights into the mutual regulation of bone formation and obesity and open new perspectives to monitor and to interfere with the dysregulated balance of adipogenesis and osteogenesis in obesity and osteoporosis.

Milk Fat Globule-Epidermal Growth Factor 8 (MFG-E8) Is a Novel Anti-inflammatory Factor in Rheumatoid Arthritis in Mice and Humans.

Milk fat globule-epidermal growth factor 8 (MFG-E8) is an anti-inflammatory glycoprotein that mediates the clearance of apoptotic cells and is implicated in the pathogenesis of autoimmune and inflammatory diseases. Because MFG-E8 also controls bone metabolism, we investigated its role in rheumatoid arthritis (RA), focusing on inflammation and joint destruction. The regulation of MFG-E8 by inflammation was assessed in vitro using osteoblasts, in arthritic mice and in patients with RA. K/BxN serum transfer arthritis (STA) was applied to MFG-E8 knock-out mice to assess its role in the pathogenesis of arthritis. Stimulation of osteoblasts with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α downregulated the expression of MFG-E8 by 30% to 35%. MFG-E8-deficient osteoblasts responded to LPS with a stronger production of pro-inflammatory cytokines. In vivo, MFG-E8 mRNA levels were 52% lower in the paws of collagen-induced arthritic (CIA) mice and 24% to 42% lower in the serum of arthritic mice using two different arthritis models (CIA and STA). Similarly, patients with RA (n = 93) had lower serum concentrations of MFG-E8 (-17%) compared with healthy controls (n = 140). In a subgroup of patients who had a moderate to high disease activity (n = 21), serum concentrations of MFG-E8 rose after complete or partial remission had been achieved (+67%). Finally, MFG-E8-deficient mice subjected to STA exhibited a stronger disease burden, an increased number of neutrophils in the joints, and a more extensive local and systemic bone loss. This was accompanied by an increased activation of osteoclasts and a suppression of osteoblast function in MFG-E8-deficient mice. Thus, MFG-E8 is a protective factor in the pathogenesis of RA and subsequent bone loss. Whether MFG-E8 qualifies as a novel biomarker or therapeutic target for the treatment of RA is worth addressing in further studies.

An exotic cause of exudative enteropathy.

PATIENT: Male, 50 FINAL DIAGNOSIS: Exudative enteropathy Symptoms: Abdominal pain • diarrhea • fever • hyponatremia • lymphadenopathy • weight loss MEDICATION: - Clinical Procedure: - Specialty: - OBJECTIVE: Unusual clinical course. BACKGROUND: Protein-losing enteropathy is a rare cause of hypoproteinemia. Erosive and non-erosive gastrointestinal diseases as well as vascular disorders that result in increased central venous pressure or mesenteric lymphatic obstruction may result in protein loss via the gastrointestinal tract. CASE REPORT: We present the case of a 50-year-old man with protein-losing enteropathy, who had profuse diarrhea, abdominal pain, lymphadenopathy, fever, and a weight loss of 10 kg in the preceding 2 months. Extensive work-up revealed infection with Giardia lamblia. We review clinical signs and symptoms, laboratory findings, and imaging studies, and discuss potential pitfalls in establishing the diagnosis. CONCLUSIONS: To the best of our knowledge, this represents one of the few published cases of intestinal giardiasis as a cause of protein-losing enteropathy in an immunocompetent adult. The diagnosis of lambliasis should be based on a combination of stool cultures and serum serology, and in cases of high clinical suspicion, an endoscopy and biopsy of the upper GI tract is recommended.

Effects of the selective glucocorticoid receptor modulator compound A on bone metabolism and inflammation in male mice with collagen-induced arthritis.

Glucocorticoids (GCs) are potent drugs to treat rheumatoid arthritis but exert adverse skeletal effects. Compound A (CpdA) is a selective GC receptor modulator with an improved risk/benefit profile in mouse models of inflammation and bone loss. Here we tested whether CpdA also exerts bone-sparing effects under proinflammatory circumstances using the collagen-induced arthritis model, a murine model of rheumatoid arthritis. CpdA decreased disease activity, paw swelling, and the paw temperature by 43%, 12%, and 7%, respectively, but was less potent than dexamethasone (DEX), which reduced these parameters by 72%, 22%, and 10%, respectively. Moreover, T cells isolated from CpdA- and DEX-treated animals were less active based on proliferation rates after challenge with type II collagen and produced smaller amounts of interferon-γ and TNF as compared with T cells from PBS-treated mice. Histological assessment of the joints confirmed the weaker potency of CpdA as compared with DEX in preventing infiltration of inflammatory cells, induction of osteoclastogenesis, and destruction of articular cartilage. Due to the lack of GC-susceptible arthritis models, we were not able to fully address the bone-sparing potential of CpdA in inflammatory conditions. Nevertheless, the bone formation marker procollagen type 1 N-terminal peptide, a surrogate marker for GC-mediated suppression of bone formation, was significantly decreased by DEX in arthritic mice but not by CpdA. Our data indicate that CpdA moderately suppresses inflammation, whereas the concurrent effects on bone remain unknown. In light of its narrow therapeutic range, CpdA may be more useful as a molecular tool for dissecting GC actions rather than a therapeutic agent.

WNT5A is induced by inflammatory mediators in bone marrow stromal cells and regulates cytokine and chemokine production.

WNT5A has recently been implicated in inflammatory processes, but its role as a bone marrow stromal cell (BMSC)-derived mediator of joint inflammation in arthritis is unclear. Here, we investigated whether inflammatory stimuli induce WNT5A in BMSC to control inflammatory responses. WNT5A levels were determined in human BMSC after stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF-α,) and in synovial cells and tissue of patients with rheumatoid arthritis (RA) and human TNF-α transgenic (hTNFtg) mice. A microarray analysis of WNT5A-treated murine osteoblasts was performed using Affymetrix gene chips. The regulation of cytokine/chemokine expression was confirmed by qPCR, ELISA, and Luminex technology in BMSC after stimulation with WNT5A or WNT5A knockdown. Relevant signaling pathways were identified using specific inhibitors. Migration of MACS-purified T lymphocytes and monocytes was assessed using the FluoroBlok system. WNT5A expression was increased threefold in BMSC after stimulation with LPS or TNF-α. Synovial fibroblasts from patients with RA showed a twofold increase of WNT5A expression compared with control cells, and its expression was highly induced in the synovial tissue of patients with RA and hTNFtg mice. Microarray analysis of WNT5A-treated osteoblasts identified cytokines and chemokines as targets. The induction of IL-1ß, IL-6, CCL2, CCL5, CXCL1, and CXCL5 by WNT5A was confirmed in BMSC and depended on the activation of the NF-κB, mitogen-activated protein (MAPK), and Akt pathways. Accordingly, knockdown of WNT5A markedly reduced the basal and LPS-induced cytokine/chemokine production. Finally, migration of monocytes and T cells toward the supernatant of WNT5A-treated BMSC was increased by 25% and 20%, respectively. This study underlines the critical role of BMSC-derived WNT5A in the regulation of inflammatory processes and suggests its participation in the pathogenesis of RA.